The goal of this proposal is to identify the key metabolic pathways utilized by osteoblasts in vivo for optimal bone formation. Recent studies have shown that modulation of metabolic pathways is a key determinant of cell fate and differentiation. For example, during activation immune T-cells engage aerobic glycolysis for cytokine production; vascular endothelial cells also utilize aerobic glycolysis during vessel sprouting. Hematopoietic stem cells, osteoblasts and osteoclasts depend on glutamine metabolism for optimal differentiation. In our preliminary studies we observed that osteoblasts during early stages of differentiation upregulate both oxidative phosphorylation and glycolysis. Differentiated osteoblasts on the other hand are highly glycolytic even under aerobic conditions, suggesting that they prefer aerobic glycolysis (Warburg effect). The substrates that are utilized by osteoblasts and the catabolic pathways that are active during osteoblast differentiation in vivo is currently an unknown in skeletal biology. In this proposal we will utilize a novel mouse model where we will overexpress MitoNEET a mitochondrial membrane protein specifically in the osteoblasts. MitoNEET over- expression should result in suppression of ?-Oxidation dependent oxidative phosphorylation and an increase in glycolysis. We will achieve this using two specific aims. 1) In specific aim 1 we will utilize preosteoblast MC3T3E1 cells to identify the mechanisms and metabolic pathways through which MitoNEET regulates osteoblast differentiation. We will generate MC3T3E1 cells infected with rAAVMitoNEET and control rAAVGFP viruses and utilize these cells to study OxPhos, Glyc and glutamynolysis. By manipulating glucose, pyruvate, fatty acids and amino acids as substrates and using specific inhibitors in vitro we can begin to analyze which metabolic pathways are essential for optimal differentiation. We will utilize novel Seahorse extracellular flux technology to study the differen pathways. 2) In specific aim 2 we will study the in vivo effects of MitoNEET over-expression specifically in osteoblasts. We will perform comprehensive skeletal phenotyping of rtTARunx2TREMitoNEET mice using micro-computed tomography (?CT) analysis and bone histomorphometry. This will identify the role of decreased OxPhos in bone acquisition. We will use metabolic cage studies to measure oxygen consumption, and energy expenditure of the mice and correlate these to the skeletal phenotype. We will use osmium tetraoxide staining to characterize marrow adiposity in the rtTARunx2TREMitoNEET mice. These experiments will provide insights into the role of ?-Oxidation dependent oxidative phosphorylation, and will begin to define the context specific nature of osteoblast bioenergetics during critical phases of differentiation.